Flow cytometric assessment of morphology, viability, and production of reactive oxygen species of Crassostrea gigas oocytes. Application to Toxic dinoflagellate (Alexandrium minutum) exposure

Abstract

The Pacific oyster Crassostrea gigas accounts for a large part of shellfish aquaculture production worldwide. Aspects of morphological and functional characteristics of oyster oocytes remain poorly documented, and traditional techniques, such as microscopic observations of shape or fertilization rate, are time and space consuming. The purpose of this study was to assess for the first time viability and reactive oxygen species (ROS) production of Pacific oyster oocytes using flow cytometry (FCM) and to apply this method to determine oocyte responses to in vitro exposure to the toxic dinoflagellate Alexandrium minutum. A culture of A. minutum caused a significant increase in oocyte ROS production, which gradually increased with the age of the culture, but viability was not affected. Effect of the supernatant of the same A. minutum culture did not cause any significant modifications of oocyte morphology, viability, or ROS level. This study confirmed that some oocyte cellular characteristics can be assessed using FCM techniques. V C 2014 International Society for Advancement of Cytometry Key terms Pacific oyster; gamete quality; ROS production; viability; flow cytometry; ecotoxicology THE Pacific oyster Crassostrea gigas has considerable commercial value worldwide, including in France. Oyster seed production relies upon gamete quality, fertilization success, and larval development. As gamete quality is the first step in the spatproduction sequence, it is necessary to assess the quality of gametes in broodstock oysters first and foremost.To date, bioassays to assess oocyte quality are limited, and those available are tedious and time-consuming. Oocyte quality can be described using morphological characteristics by microscopy (1). Although microscope observations are unavoidable and useful to validate flow cytometry (FCM), assays based on microscope observations often have little predictive value and can be subjective according to each observer. Determinations of biochemical content (proteins, carbohydrates, and lipids) have been used as criteria of oocyte quality (2-4). More recently, proteomic profiles of oocytes were proposed as quality estimators (5,6). All of these biochemical assays, however, are fairly tedious to perform and provide only retrospective information. Fertilization rate, hatching rate, and larval development are used as complementary end points to evaluate oocyte quality (7-10); however, as these processes depend also upon spermatozoa quality, variability and inconsistency from one experiment to another often are encountered.

The dinoflagellate Alexandrium minutum affects development of the oyster Crassostrea gigas, through parental or direct exposure

“…Gametes were collected at T8 in the remaining control and exposed animals (45 oysters per treatment, 15 oysters per tank) by stripping the gonad, and pooling gametes of oysters from the same tank, for each sex. Oocytes were suspended in FSW, filtered in a 100-µm sieve, and oocyte concentrations were determined by flow cytometry (FCM) according to Le Goïc et al (2014). Oocytes were transferred in 2 mL tubes for PST quantification and biochemical composition measurements (2 × 10 6 oocytes for each analysis, stored at -20 °C), and for RNA analyses (4 × 10 6 oocytes, stored in liquid nitrogen).…”

“…the gills (Borcier et al, Castrec et al, 2018) or cells, e.g. gametes (Le Goïc et al, 2014). These BECs could be cytotoxic to the velum, the feeding and swimming organ of the larvae, thereby reducing energy uptake and subsequent larval growth.…”

Gametogenesis-Related Fluctuations in Ovothiol Levels in the Mantle of Mussels from Different Estuaries: Fighting Oxidative Stress for Spawning in Polluted Waters

“…In the case of spermatogenesis, this could serve as a protective measure along differentiation but specially in oocytes, where it could have additional functions. Broadcast spawners, such as marine bivalves, spawn their gametes in the environment where exposure to environmental variables and pollutants may increase the production of ROS, as it has been proven in oyster oocytes exposed in vitro to dispersed oil [27] or to toxic dinoflagellates [28]. Similarly, fertilization and early embryo cell cleavage is characterized by an increase of H 2 O 2 production in marine invertebrates [1,4,9] and antioxidants accumulated in oocytes are bound to counteract its pro-oxidant effect.…”

Ultrafiltration for environment safety in shellfish production: Removal of oyster gametes in hatchery effluents

“…corresponds to oocytes or spermatozoa (Le Goic et al, 2014;Vignier et al, 2017). The gamete samples were analysed by flow cytometry on a Partec Cyflow Space flow cytometer to determine the concentration.…”

“…This solution was then analysed after 10 min in the dark (Le Goic et al, 2014). Spermatozoa viability was determined using the same method but with SYBR-14 instead of SYBR-Green.…”